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The effect of a depot injection of recombinant bovine somatotropin on follicular development and embryo yield in superovulated Holstein heifers
Theriogenology
40: 1003-I
013,1993
THE EFFECT OF A DEPOT INJECTION OF RECOMBINANT BOVINE SOMATOTROPIN ON FOLLICLJLAR DEVELOPMENT AND EMBRYO YIELD IN SUPEROVULATED HOLSTEIN HEIFERS L.F. Kuehner,l
D. Rieger,la
J.S. Walton,* X. Zhao 2 and W.H. Johnson3
Departments of IBiomedical Sciences, *Animal and Poultry Science and 3Population Medicine, University of Guelph Guelph, Ontario, Canada, NlG 2Wl Received for publication: &Zy 3, 1992 Accepted: JULY 29, 1993 ABSTRACT Superovulated Holstein heifers (n = 32) were given a depot injection of 500 mg recombinant bovine somatotropin (rBST) or vehicle at Day 4 of the estrous cycle (7 days before the first FSH injection); at Day 11, coincidentally with the first FSH injection; or at Day 15, the time of artificial insemination. Embryos were collected nonsurgically, and the number of corpora lutea was counted by ultrasonography at Day 7 after insemination. Blood samples were taken every second day, from Day 2 of the superovulatory cycle until the day of embryo collection, and were analyzed for progesterone, somatotropin and insulin-like growth factor-l (IGF-1). Somatotropin-treated heifers at Day 11 had a significantly higher mean number of corpora lutea than the controls (18.1 vs 13.4; P I 0.05). Day 4 treatment tended to increase the mean number of corpora lutea (15.4; P < O.lO), and significantly increased the overall percentage of transferable embryos (74.6 vs 58.6%; P $ 0.01). In the control animals, plasma IGF-1 was uncorrelated to somatotropin (P > 0.63), but it was negatively correlated with progesterone (P 5 O.Ol), suggesting that IGF-1 production in the superovulated heifer may be related to ovarian development. Key words:
superovulation,
cattle, rBST, IGF-1
Acknowledgements The authors thank Dr. Keith A. MacMillan of Monsanto Canada, Inc., for providing the recombinant bovine somatotrophin; Robert Beriault, Chris Gwyn and Suzanne Manning for management of the animals; and Michelle Goodwin and George Werchola for assistance with the hormone The antiserum and highly purified pituitary hormone for the assays. somatotrophin assay was provided by Dr. A.F. Par-low, Pituitary Hormones and Antisera Center, Harbor-UCLA Medical Center, Torrance, CA, USA. Drs. L.E. Underwood and J.J. Van Wyk, U.S. National Pituitary Agency Financial support was provided the antiserum for the IGF-1 assay. provided by the NSERC Canada Strategic Grants Program and the Ontario Ministry of Agriculture and Food. aReprint requests.
Copyright
0 1993 Butterworth-Heinemann
1004
Theriogenology
INTRODUCTION Growth factors, particularly insulin-like growth factor-l (IGF-I), have been shown to stimulate granulosa and theta cell differentiation, steroidogenesis and oocyte maturation in a variety of species (l), and are therefore considered to modulate the effects of gonadotrophins on ovarian follicular development (2). In addition to their effects on follicular development, growth factors, including IGF-1, are known to have wideranging stimulatory or facilitatory effects on the metabolism, growth and differentiation of early-stage mammalian embryos (3-5). In women, ovarian stimulation with human menopausal gonadotrophin (hMG) results in increased endogenous somatotropin secretion (6), and the ovarian response is directly related to the clonidinestimulated somatotropin release (7). The secretion of somatotropin in response to an arginine challenge has similarly been shown to be greater in regularly cycling, than in anovulatory, women (8). The supply of endogenous somatotropin appears to be insufficient for an optimal response to ovarian stimulation because follicular development is significantly improved by cotreatment with recombinant human somatotropin (9-14). In superovulated heifers, twice-daily injections of recombinant bovine somatotropin (rBST) given concomitantly with FSH injections produced an increased ovulatory response, but had no significant effect on the number of transferable embryos (15). Herrler et al. (16) used a depot injection of rBST given 5 days before a superovulatory PMSG injection and obtained an increased number of transferable embryos, but the mean number of transferable embryos produced by the control group (1.9) was quite low. In this study, a before FSH treatment follicles, at the time of the rescue from atresia artificial insemination development.
Animals A total of 32 were used between maintenance ration monitored for estrus fined as Day 0 of the while 9 others were
depot injection of rBST was administered at 7 days to determine the effects on early growth of antral initiation of FSH treatment to determine the effect on and final growth of antral follicles, or at the time of (AI) to determine the effect on early embryo
MATERIALS
AND METHODS
Holstein heifers, ranging in age from 18 to 43 months, January and November, 1991. The heifers were fed a of hay and grain, allowed water ad libitum, and were twice daily. The day of the first detected estrus was deestrous cycle or gestation. Nine animals were used twice, used 3 times. All the heifers had at least 2 successive
1005
Theriogenology
estrous cycles, 19-22 days in length before the first, or between treatments. Superovulatory
Treatments
successive,
and Embryo Collection
For each of 12 trials, groups of 4 to 6 luteal-phase animals were randomly selected from the experimental herd and were given 2 injections of 0.5 mg i.m. of cloprostenolb (PG) to synchronize estrus. A superovulatory regimen was started at Day 11 of the ensuing cycle, consisting of twice-daily intramuscular injections in decreasing dosages of porcine follicle-stimulating hormonec (FSH) for 4 days (5.25, 5.25, 4.38, 4.38, 3.50, 3.50, 2.63, 2.63 mg, total dose = 31.5 mg) together with 2 injections of 0.5 mg i.m. of PG with the fifth and sixth injections of FSH. All animals were artificially inseminated twice daily throughout estrus, beginning at 48 hours after the first PG injection, using frozen-thawed semen from the same ejaculate. The embryos were collected by a nonsurgical flushing of the uterus at Day 7, isolated from the flushing fluid, and graded according to morphological criteria (17). Only embryos graded good or excellent were considered to be of transferable quality. The number of corpora lutea was estimated at the time of embryo collection by real-time transrectal ultrasonography using a linear-array scanner equipped with a 5 MHz probe! rBST Treatment The animals were randomly assigned to receive a single depot injection of 500 mg S.C. of rBSTe or of vehicle behind the shoulder at Day 4 (7 days prior to the first FSH injection); Day 11 (time of first FSH injection); or at Day 15 (time of first AI). Fifteen rBST and 5 vehicle treatments were given at each treatment period, for a total of 60 superovulations. The number of treatments given on the first, second or third superovulation in each treatment group was 9,3 and 3 for the controls; 8,5 and 2 for Day 4; 8,5 and 2 for Day 11; and 7,5 and 3 for Day 15. Blood Sampling and Hormone Assays Blood samples were taken by coccygeal venipuncture into heparinized tubes every second day from Day 2 of the synchronized cycle until the day of embryo collection (Day 7 of gestation). The blood was centrifuged immediately after collection, and the plasma was frozen and stored at -2OOC All samples taken during the first 32 until assayed for hormone content.
b c d e
Estrumate, Coopers Agropharm, Willowdale, Ontario, Canada. Folltropin, Vetrepharm, London, Ontario, Canada. Equisonics Inc., Elk Grove, IL, USA. Sometribove, Monsanto Canada Inc., Mississuaga, Ontario, Canada.
1006
Theriogenology
superovulations 00 controls, 7 Day-4 rBST, 8 Day-11 rBST, and 7 Day-15 rBST) were analyzed for progesterone, somatotropin, and IGF-I. Progesterone content only was determined in the last samples (day of embryo collection) for the remaining superovulations. Plasma concentrations of somatotropin and IGF-1 were determined in single radioimmunoassays, as described by Elsasser et al. (18) and previously validated in our institution (19). The assay sensitivities and intra-assay coefficients of variation were 0.25 ngfml and 18.9% for somatotropin, and 7.5 ng/ml and 4.7% for IGF-1. Plasma concentrations of progesterone were determined in a single radioimmunoassay, as previously described (20). The assay sensitivity and intra-assay coefficient of variation were 0.125 ng/ml and 11.5%. Statistical Analysis All hormone concentrations and numbers of corpora lutea were logtransformed for statistical analysis to provide homogeneity of variance. The plasma concentrations of progesterone, somatotropin and IGF-1 were compared among treatment groups by analysis of variance for repeated measures (reagent X day). The association between plasma concentrations of somatotropin and IGF-1 in the rBST treated animals were evaluated by Pearson’s product-moment correlations. The numbers of corpora lutea, total and transferable embryos, and the percentage of transferable embryos were compared among groups by one-way analysis of variance, followed by Duncan’s Multiple Range tests. Comparisons of the overall percentages of transferable embryos between the control group and the rBST-treated groups were made by Chi-square analyses. For the control group only, the effect of day on plasma concentrations of somatotropin, IGF-1 and progesterone was evaluated by one-way analysis between all 3 of variance for repeated measures, and the associations hormones were evaluated by Pearson’s product-moment correlations. RESULTS There were no significant differences among the 3 vehicle-treated groups for any of the measured variables and the data were therefore pooled. The back-transformed Ieast-squares means of plasma concentrations of progesterone, somatotropin and IGF-I in the rBST depot-treated groups are As expected, plasma progesterone concentrations shown in Figure 1. decreased after PG and increased markedly between Days 1 and 7 of gestation in all the groups. There was a highly significant effect of day (I’ 5 0.001) on plasma progesterone concentration, but no significant effect of treatment group or group X day interaction was observed.
1007
Theriogenology
:
30
PG
$
., __ :. ,..,..,..:;:::.I :: m;
AI 1
.
,.
c
-?
Day of the Estrous Cycle or Gestation Figure 1 Mean plasma progesterone, somatotropin and IGF-1 concentrations in superovulated Holstein heifers administered a single depot injection of vehicle (n = 10) or 500 mg of rBST on Day 4 (n = 7), on Day 11 (n = 81, or on Day 15 (n = 7).
1008
Theriogenology
Plasma concentrations of somatotropin and IGF-1 increased markedly within 2 days following rBST treatment, and remained above control values for as long as 12 days in the treated groups. There was a significant effect of treatment group (P I 0.05) and a highly significant effect of day (P < 0.001) and group X day interaction (P < 0.001) for the plasma concentrations of both somatotropin and IGE-1. In all the rBST-treated animals, there was a highly significant positive correlation between plasma somatotropin and IGF-1 (r = 0.30, P IO.001). The mean numbers of corpora lutea and embryos collected at Day 7 are shown in Table 1. One animal given rBST at Day 4 was subsequently found to have a cystic follicle and was removed from the study. Mechanical difficulties were encountered in attempting to flush 1 animal from the Day-4 rBST group and 1 from the control group; therefore no embryo data were available for No embryos were recovered from 1 control heifer and these 2 animals. therefore the percentage transferable embryos (Table 2) could not be calculated for that collection. Compared with the control group, the mean number of corpora lutea was significantly higher in the Day-11 rBST-treated group (P .%0.05), and it tended to be higher in the Day-4 rBST-treated group (P I 0.10). The number of corpora lutea was not significantly different among the 3 rBST-treated groups, and there were no differences in the total number of embryos recovered among the treatment groups. All the rBST-treated groups yielded approximately 1 more transferable embryo per collection than did the controls, but there were no significant differences among the treatment The percentage of transferable embryos did not differ among groups. treatment groups when compared on a per collection basis, but the overall percentage of transferable embryos (Table 2) was significantly higher in the Day-4 rBST-treated group than in the controls (P S 0.01). Table 1. The effect of a single 500-mg depot injection of rBST on the mean numbers (+ SEM) of corpora lutea and embryos collected from superovulated heifers. The number of collections represented in each mean are shown in parentheses. Group
Con trofs rBST at Day 4 rBST at Day 11 rBST at Day 15
Corpora lutea 13.4 15.4 18.1 16.5
+ 1.6a k 1.1ab f 1.3b f 2.4ab
Transferable embryos
Total embryos (15) (14) (15) (15)
10.0 9.1 11.2 11.1
* 1.5 i: 2.0 It 1.5 i: 2.2
(14) (13) (15) (15)
5.9 6.8 7.1 7.0
2 1.4 + 1.7 zlz1.4 -e 1.7
a,bMeans with different superscripts are significantly different (P 5 0.05).
(14)
(13) (15) (15)
Theriogenology
1009
Table 2. The effect of a single 500-mg depot injection of rBST on the mean per collection (k SEM) and the overall percentage of transferable embryos collected from superovulated Holstein heifers. Numbers in parentheses are transferable/total embryos collected from each group. Group
n
Controls rBST at Day 4 rBST at Day 12 rBST at Day 15
Per collection % transferable
13 13 15 15
59.0 67.8 61.8 55.2
+ k i +
9.6 9.2 7.4 7.0
Overall % transferable 58.6 74.6a 63.1 62.9
(f&2/140) (88/118) (106/X8) (205,067)
aP 2 0.01 compared with controls. Across all collections, there was a highly significant correlation of the number of corpora lutea with plasma progesterone on the day of embryo collection (r-=0.48; I? i 0.002) and with the total number of embryos recovered (1:= 0.37; P < 0.01). I’G
AI
r 170
I =: - 6 - 160
150 & 5
z - 140 2
- 130
2
4
6
8
10 12 14 1 3 5 Day of the Estrous Cycle or Gestation
Figure 2. Mean of plasma progesterone and IGF-1 superovulated control Holstein heifers (n = 40).
7
$ E:
120
concentrations
in
1010
Theriogenology
The back-transformed least-squares means of plasma concentrations of progesterone and IGF-1 in the control group are shown in Figure 2. There was a significant effect of day (P < 0.05) on the concentrations of progesterone and IGF-1 but not of somatotropin, over the sampling period. There was no correlation between plasma somatotropin and IGF-1 (r = 0.05, P > 0.76) or between progesterone and somatotropin (r = 0.04, P > 0.631, but there was a significant negative correlation between progesterone and IGF-1 (r = -0.26, P I 0.01). DISCUSSION The significant increase in the number of corpora lutea in superovulated heifers administered 500 mg of rBST depot on Day 11, coincidentally with the first FSH injection, is consistent with our previous observation that multiple (twice-daily) 20-mg injections of rBST during FSH treatment improves the superovulatory response (15). The tendency toward an increased number of corpora lutea in the animals given the rBST depot at Day 4 is similar to the moderate, nonsignificant increase observed in cows given a 640-mg depot of rBST, 5 days before treatment with PMSG (16). These observations suggest that rBST, either directly or through IGF-1, improves the ability of subordinate follicles to develop in response to exogenous gonadotrophins in heifers, as has been shown in women (9-14). However, the fact that treatment with the depot on Day 15 (at the time of AI) had no effect on the number of corpora lutea suggests that rBST did not directly affect ovulation or luteinization. Treatment with the rBST depot had no statistically significant effect on the total number of embryos or on the number of transferable embryos, consistent with our previous observations using twice-daily injections of rBST (15), but all 3 rBST-treated groups yielded approximately one more transferable embryo per collection than did the controls. Moreover, the overall percentage of transferable embryos was significantly increased (P I 0.01) in the animals administered the rBST depot on Day 4. This may suggest that somatotropin or IGF-1 is particularly important for the early development of the antral follicle and the oocyte. Gong et al. (21) reported that daily injections of rBST over 2 estrous cycles doubles the number of small antral follicles in otherwise untreated heifers, suggesting that somatotropin or IGF-1 can affect follicular development at an even earlier stage. Although our experiment was designed to test only the effects of rBST on the superovulatory response, the inverse relationship that we observed between plasma concentrations of progesterone and IGF-1 in the control animals was striking, and perhaps of fundamental interest. This relationship may suggest that the secretion of IGF-1 is related to one or more factors
associated with ovarian development in the superovulated heifer, but that mechanism is not clear. Plasma concentrations of IGF-1 decrease significantly within 24 hours after ovariectomy in beef-cows (22) and cross-breed heifers (23), and increase significantly within 14 hours after treatment of ovariectomized cows with estradiol (22). It is therefore possible that the inverse relationship between IGF-I and progesterone reflected the changes in circulating estradiol during follicular development. However, further investigation is required to determine the exact mechanism and physiological significance of changes in 1GF-l secretion during the estrous cycle in cattle. In conclusion, treatment of superovulated heifers with a 500-mg depot injection of rBST on Day 11, coincidentally with the first FSH injection, produced a significant increase in the ovulatory response. Treatment with the depot on Day 4,7 days before the first FSH injection, tended to increase the ovulatory response and significantly increased the percentage of transferable In the control animals, plasma concentrations of IGF-1 were embryos. negatively correlated with progesterone, but were statistically unrelated to somatotropin, suggesting that IGF-1 secretion in the superovulated heifer may be related to some factor associated with follicular development, possibly to circulating estradiol.
1.
Hammond, J.M., Mondschein, J.S., Samaras, S.E., Smith, S.A. and Hagen, D.R. The ovarian insulin-like growth factor system. J. Reprod. Fertil. 43 (Suppl.):199-208 (1991).
2.
Dorrington, J.H., Bendell, J.J., Chuma, A. and Lobb, D.K. Actions of growth factors in the follicle. J. Steroid Biochem. 27:405-411 (1987).
3.
Defining the roles of growth factors during early Riazino, A. mammalian development. In: Bavister, B. D. (ed) The Mammalian Preimplantation Embryo. Regulation of Growth and Differentiation in Vitro. Plenum Press, New York, 1987, pp. 151-174.
4.
Slack, J.M.W. Peptide regulatory Lancet 1;:1312-1315 (1989).
5.
Growth Whitman, M. and MeIton, D.A. embryogenesis. Ann. Rev. Cell Biol. 393-117 (1989).
6.
Effect of Wilson, E.E., Word, R.A., Byrd, W. and Carr, B.R. superovulation with human menopausal gonadotropins on growth hormone levels in women. J. Clin, Endocrinol. Metab. 73:511-515 (1991).
factors in embryonic
development.
factors
in
early
1012
Theriogenoiogy
7
Menashe, Y., Lunenfeld, B., Pariente, C., Frenkel, Y. and Mashiach, S. Can growth hormone increase, after clonidine administration, predict the dose of human menopausal hormone needed for induction of ovulation. Fertil. Steril. %:432-435 (1990).
8.
Oyesen, I’., Moller, Jorgensen,, J.O.L. insulin-like growth women with regular
9.
Homburg, R., West, C. and Jacobs, H.S. Controlled trial of co-treatment with growth hormone and gonadotrophins for induction of ovulation. J. Endocrinol. 119 ~Suppl.):l44 abstr. (1988).
10.
Blumenfeld, Z. and Lunenfeld, B. The potentiating’ effect of growth hormone on follicle stimulation with human menopausal gonadotropin in a panhypopituitary patient. @:328-331 (1989).
11.
Matson, P.L., Ibrahim, Z.H.Z., Morris, I.D., Sun, J.G. and Lieberman, B.A. Effect of biosynthetic human growth hormone upon follicular growth in women undergoing ovarian stimulation prior to in-vitro fertilisation and embryo transfer. J. Reprod. Fertil. Abstr. Ser. 4:17 abstr. (1989).
12.
Homburg, R., West, C., Torresani, T. and Jacobs, H.S. Cotreatment with human growth hormone and gonadotropins for induction of ovulation: a controlled clinical trial. Fertil. Steril. 3:254-260 (1990).
13.
Homburg, R., West, ‘C., Torresani, T. and Jacobs, H.S. A comparative study of single-dose growth hormone therapy as an adjuvant to gonadotrophin treatment for ovulation induction. Clin. Endocrinol. 32:78X-785 (1990).
14.
Ibrahim, Z.H.Z., Matson, P.L., Buck, P. and Lieberman, B.A. The use of biosynthetic human growth hormone to augment ovulation induction with buserelin acetate/human menopausal gonadotropin in women with a poor ovarian response. Fertil. Steril. z:202-204 (1991).
15.
Rieger, D., Walton, J.S., Goodwin, M.L. and Johnson, W.H. The effect of co-treatment with recombinant bovine somatotrophin on plasma progesterone concentration and numbers of embryos collected from superovulated Holstein heifers. Theriogenology %:863-868 (1991).
16.
Herrler, A., Farries, E. and Niemann, H. A trial to stimulate insulin like growth factor 1 levels to improve superovulatory response in dairy cows. 33:248 abstr. (1990).
J., Moller, N., Christiansen, J.S., Orskov, H. and Growth hormone secretory capacity and serum factor-I levels in primary Infertile, anovulatory menses. Fertil. Steril. 57:97-101 (1992).
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Theriogenology
17.
Elsden, R.P., Nelson, L.D. and Seidel, G.E. Superovulating cows with follicle stimulating hormone and pregnant mare’s serum gonadotrophin. Theriogenology 2:17-26 (1978).
18.
Elsasser, T.H., Rumsey, T.S. and Hammond, A.C. Influence of diet on basal and growth hormone-stimulated plasma concentrations of IGF-1 in beef cattle. a:128-141 (1989).
19.
Groenewegen, P.P., McBride, B.W., Burton, J.H. and Elsasser, T.H. Effect of bovine somatotropin on the growth rate, hormone profiles and carcass composition of Holstein bull calves. Dom. Anim. Endocrinol. 2:43-54 (1990).
20.
de Boer, G., Etches, R.J. and radioimmunoasay for progesterone Sci. @:783-786 (1980).
21.
Gong, J.G., Bramley, T. and Webb, R. The effect of recombinant bovine somatotropin on ovarian function in heifers: follicular populations and peripheral hormones. Biol. Reprod. a:941-949 (1991).
22.
Richards, M.W., Wettemann, RI’., Spicer, Nutritional anestrus in beef cows: effects ovariectomy on serum luteinizing hormone factor-l. Biol. Reprod. @:961-966 (1991).
23.
Spicer, L.J., Crow, M.A., Prendiville, D. J., Goulding, D. and Enright, Systemic but not intraovarian concentrations of insulin-like W.J. growth factor-l are affected by short-term fasting. Biol. Reprod. &:920925 (1992).
Walton, J.S. A solid-phase in bovine plasma. Can. J. Anim.
L.J. and Morgan, G.L. of body condition and and insulin-like growth
40: 1003-I
013,1993
THE EFFECT OF A DEPOT INJECTION OF RECOMBINANT BOVINE SOMATOTROPIN ON FOLLICLJLAR DEVELOPMENT AND EMBRYO YIELD IN SUPEROVULATED HOLSTEIN HEIFERS L.F. Kuehner,l
D. Rieger,la
J.S. Walton,* X. Zhao 2 and W.H. Johnson3
Departments of IBiomedical Sciences, *Animal and Poultry Science and 3Population Medicine, University of Guelph Guelph, Ontario, Canada, NlG 2Wl Received for publication: &Zy 3, 1992 Accepted: JULY 29, 1993 ABSTRACT Superovulated Holstein heifers (n = 32) were given a depot injection of 500 mg recombinant bovine somatotropin (rBST) or vehicle at Day 4 of the estrous cycle (7 days before the first FSH injection); at Day 11, coincidentally with the first FSH injection; or at Day 15, the time of artificial insemination. Embryos were collected nonsurgically, and the number of corpora lutea was counted by ultrasonography at Day 7 after insemination. Blood samples were taken every second day, from Day 2 of the superovulatory cycle until the day of embryo collection, and were analyzed for progesterone, somatotropin and insulin-like growth factor-l (IGF-1). Somatotropin-treated heifers at Day 11 had a significantly higher mean number of corpora lutea than the controls (18.1 vs 13.4; P I 0.05). Day 4 treatment tended to increase the mean number of corpora lutea (15.4; P < O.lO), and significantly increased the overall percentage of transferable embryos (74.6 vs 58.6%; P $ 0.01). In the control animals, plasma IGF-1 was uncorrelated to somatotropin (P > 0.63), but it was negatively correlated with progesterone (P 5 O.Ol), suggesting that IGF-1 production in the superovulated heifer may be related to ovarian development. Key words:
superovulation,
cattle, rBST, IGF-1
Acknowledgements The authors thank Dr. Keith A. MacMillan of Monsanto Canada, Inc., for providing the recombinant bovine somatotrophin; Robert Beriault, Chris Gwyn and Suzanne Manning for management of the animals; and Michelle Goodwin and George Werchola for assistance with the hormone The antiserum and highly purified pituitary hormone for the assays. somatotrophin assay was provided by Dr. A.F. Par-low, Pituitary Hormones and Antisera Center, Harbor-UCLA Medical Center, Torrance, CA, USA. Drs. L.E. Underwood and J.J. Van Wyk, U.S. National Pituitary Agency Financial support was provided the antiserum for the IGF-1 assay. provided by the NSERC Canada Strategic Grants Program and the Ontario Ministry of Agriculture and Food. aReprint requests.
Copyright
0 1993 Butterworth-Heinemann
1004
Theriogenology
INTRODUCTION Growth factors, particularly insulin-like growth factor-l (IGF-I), have been shown to stimulate granulosa and theta cell differentiation, steroidogenesis and oocyte maturation in a variety of species (l), and are therefore considered to modulate the effects of gonadotrophins on ovarian follicular development (2). In addition to their effects on follicular development, growth factors, including IGF-1, are known to have wideranging stimulatory or facilitatory effects on the metabolism, growth and differentiation of early-stage mammalian embryos (3-5). In women, ovarian stimulation with human menopausal gonadotrophin (hMG) results in increased endogenous somatotropin secretion (6), and the ovarian response is directly related to the clonidinestimulated somatotropin release (7). The secretion of somatotropin in response to an arginine challenge has similarly been shown to be greater in regularly cycling, than in anovulatory, women (8). The supply of endogenous somatotropin appears to be insufficient for an optimal response to ovarian stimulation because follicular development is significantly improved by cotreatment with recombinant human somatotropin (9-14). In superovulated heifers, twice-daily injections of recombinant bovine somatotropin (rBST) given concomitantly with FSH injections produced an increased ovulatory response, but had no significant effect on the number of transferable embryos (15). Herrler et al. (16) used a depot injection of rBST given 5 days before a superovulatory PMSG injection and obtained an increased number of transferable embryos, but the mean number of transferable embryos produced by the control group (1.9) was quite low. In this study, a before FSH treatment follicles, at the time of the rescue from atresia artificial insemination development.
Animals A total of 32 were used between maintenance ration monitored for estrus fined as Day 0 of the while 9 others were
depot injection of rBST was administered at 7 days to determine the effects on early growth of antral initiation of FSH treatment to determine the effect on and final growth of antral follicles, or at the time of (AI) to determine the effect on early embryo
MATERIALS
AND METHODS
Holstein heifers, ranging in age from 18 to 43 months, January and November, 1991. The heifers were fed a of hay and grain, allowed water ad libitum, and were twice daily. The day of the first detected estrus was deestrous cycle or gestation. Nine animals were used twice, used 3 times. All the heifers had at least 2 successive
1005
Theriogenology
estrous cycles, 19-22 days in length before the first, or between treatments. Superovulatory
Treatments
successive,
and Embryo Collection
For each of 12 trials, groups of 4 to 6 luteal-phase animals were randomly selected from the experimental herd and were given 2 injections of 0.5 mg i.m. of cloprostenolb (PG) to synchronize estrus. A superovulatory regimen was started at Day 11 of the ensuing cycle, consisting of twice-daily intramuscular injections in decreasing dosages of porcine follicle-stimulating hormonec (FSH) for 4 days (5.25, 5.25, 4.38, 4.38, 3.50, 3.50, 2.63, 2.63 mg, total dose = 31.5 mg) together with 2 injections of 0.5 mg i.m. of PG with the fifth and sixth injections of FSH. All animals were artificially inseminated twice daily throughout estrus, beginning at 48 hours after the first PG injection, using frozen-thawed semen from the same ejaculate. The embryos were collected by a nonsurgical flushing of the uterus at Day 7, isolated from the flushing fluid, and graded according to morphological criteria (17). Only embryos graded good or excellent were considered to be of transferable quality. The number of corpora lutea was estimated at the time of embryo collection by real-time transrectal ultrasonography using a linear-array scanner equipped with a 5 MHz probe! rBST Treatment The animals were randomly assigned to receive a single depot injection of 500 mg S.C. of rBSTe or of vehicle behind the shoulder at Day 4 (7 days prior to the first FSH injection); Day 11 (time of first FSH injection); or at Day 15 (time of first AI). Fifteen rBST and 5 vehicle treatments were given at each treatment period, for a total of 60 superovulations. The number of treatments given on the first, second or third superovulation in each treatment group was 9,3 and 3 for the controls; 8,5 and 2 for Day 4; 8,5 and 2 for Day 11; and 7,5 and 3 for Day 15. Blood Sampling and Hormone Assays Blood samples were taken by coccygeal venipuncture into heparinized tubes every second day from Day 2 of the synchronized cycle until the day of embryo collection (Day 7 of gestation). The blood was centrifuged immediately after collection, and the plasma was frozen and stored at -2OOC All samples taken during the first 32 until assayed for hormone content.
b c d e
Estrumate, Coopers Agropharm, Willowdale, Ontario, Canada. Folltropin, Vetrepharm, London, Ontario, Canada. Equisonics Inc., Elk Grove, IL, USA. Sometribove, Monsanto Canada Inc., Mississuaga, Ontario, Canada.
1006
Theriogenology
superovulations 00 controls, 7 Day-4 rBST, 8 Day-11 rBST, and 7 Day-15 rBST) were analyzed for progesterone, somatotropin, and IGF-I. Progesterone content only was determined in the last samples (day of embryo collection) for the remaining superovulations. Plasma concentrations of somatotropin and IGF-1 were determined in single radioimmunoassays, as described by Elsasser et al. (18) and previously validated in our institution (19). The assay sensitivities and intra-assay coefficients of variation were 0.25 ngfml and 18.9% for somatotropin, and 7.5 ng/ml and 4.7% for IGF-1. Plasma concentrations of progesterone were determined in a single radioimmunoassay, as previously described (20). The assay sensitivity and intra-assay coefficient of variation were 0.125 ng/ml and 11.5%. Statistical Analysis All hormone concentrations and numbers of corpora lutea were logtransformed for statistical analysis to provide homogeneity of variance. The plasma concentrations of progesterone, somatotropin and IGF-1 were compared among treatment groups by analysis of variance for repeated measures (reagent X day). The association between plasma concentrations of somatotropin and IGF-1 in the rBST treated animals were evaluated by Pearson’s product-moment correlations. The numbers of corpora lutea, total and transferable embryos, and the percentage of transferable embryos were compared among groups by one-way analysis of variance, followed by Duncan’s Multiple Range tests. Comparisons of the overall percentages of transferable embryos between the control group and the rBST-treated groups were made by Chi-square analyses. For the control group only, the effect of day on plasma concentrations of somatotropin, IGF-1 and progesterone was evaluated by one-way analysis between all 3 of variance for repeated measures, and the associations hormones were evaluated by Pearson’s product-moment correlations. RESULTS There were no significant differences among the 3 vehicle-treated groups for any of the measured variables and the data were therefore pooled. The back-transformed Ieast-squares means of plasma concentrations of progesterone, somatotropin and IGF-I in the rBST depot-treated groups are As expected, plasma progesterone concentrations shown in Figure 1. decreased after PG and increased markedly between Days 1 and 7 of gestation in all the groups. There was a highly significant effect of day (I’ 5 0.001) on plasma progesterone concentration, but no significant effect of treatment group or group X day interaction was observed.
1007
Theriogenology
:
30
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., __ :. ,..,..,..:;:::.I :: m;
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Day of the Estrous Cycle or Gestation Figure 1 Mean plasma progesterone, somatotropin and IGF-1 concentrations in superovulated Holstein heifers administered a single depot injection of vehicle (n = 10) or 500 mg of rBST on Day 4 (n = 7), on Day 11 (n = 81, or on Day 15 (n = 7).
1008
Theriogenology
Plasma concentrations of somatotropin and IGF-1 increased markedly within 2 days following rBST treatment, and remained above control values for as long as 12 days in the treated groups. There was a significant effect of treatment group (P I 0.05) and a highly significant effect of day (P < 0.001) and group X day interaction (P < 0.001) for the plasma concentrations of both somatotropin and IGE-1. In all the rBST-treated animals, there was a highly significant positive correlation between plasma somatotropin and IGF-1 (r = 0.30, P IO.001). The mean numbers of corpora lutea and embryos collected at Day 7 are shown in Table 1. One animal given rBST at Day 4 was subsequently found to have a cystic follicle and was removed from the study. Mechanical difficulties were encountered in attempting to flush 1 animal from the Day-4 rBST group and 1 from the control group; therefore no embryo data were available for No embryos were recovered from 1 control heifer and these 2 animals. therefore the percentage transferable embryos (Table 2) could not be calculated for that collection. Compared with the control group, the mean number of corpora lutea was significantly higher in the Day-11 rBST-treated group (P .%0.05), and it tended to be higher in the Day-4 rBST-treated group (P I 0.10). The number of corpora lutea was not significantly different among the 3 rBST-treated groups, and there were no differences in the total number of embryos recovered among the treatment groups. All the rBST-treated groups yielded approximately 1 more transferable embryo per collection than did the controls, but there were no significant differences among the treatment The percentage of transferable embryos did not differ among groups. treatment groups when compared on a per collection basis, but the overall percentage of transferable embryos (Table 2) was significantly higher in the Day-4 rBST-treated group than in the controls (P S 0.01). Table 1. The effect of a single 500-mg depot injection of rBST on the mean numbers (+ SEM) of corpora lutea and embryos collected from superovulated heifers. The number of collections represented in each mean are shown in parentheses. Group
Con trofs rBST at Day 4 rBST at Day 11 rBST at Day 15
Corpora lutea 13.4 15.4 18.1 16.5
+ 1.6a k 1.1ab f 1.3b f 2.4ab
Transferable embryos
Total embryos (15) (14) (15) (15)
10.0 9.1 11.2 11.1
* 1.5 i: 2.0 It 1.5 i: 2.2
(14) (13) (15) (15)
5.9 6.8 7.1 7.0
2 1.4 + 1.7 zlz1.4 -e 1.7
a,bMeans with different superscripts are significantly different (P 5 0.05).
(14)
(13) (15) (15)
Theriogenology
1009
Table 2. The effect of a single 500-mg depot injection of rBST on the mean per collection (k SEM) and the overall percentage of transferable embryos collected from superovulated Holstein heifers. Numbers in parentheses are transferable/total embryos collected from each group. Group
n
Controls rBST at Day 4 rBST at Day 12 rBST at Day 15
Per collection % transferable
13 13 15 15
59.0 67.8 61.8 55.2
+ k i +
9.6 9.2 7.4 7.0
Overall % transferable 58.6 74.6a 63.1 62.9
(f&2/140) (88/118) (106/X8) (205,067)
aP 2 0.01 compared with controls. Across all collections, there was a highly significant correlation of the number of corpora lutea with plasma progesterone on the day of embryo collection (r-=0.48; I? i 0.002) and with the total number of embryos recovered (1:= 0.37; P < 0.01). I’G
AI
r 170
I =: - 6 - 160
150 & 5
z - 140 2
- 130
2
4
6
8
10 12 14 1 3 5 Day of the Estrous Cycle or Gestation
Figure 2. Mean of plasma progesterone and IGF-1 superovulated control Holstein heifers (n = 40).
7
$ E:
120
concentrations
in
1010
Theriogenology
The back-transformed least-squares means of plasma concentrations of progesterone and IGF-1 in the control group are shown in Figure 2. There was a significant effect of day (P < 0.05) on the concentrations of progesterone and IGF-1 but not of somatotropin, over the sampling period. There was no correlation between plasma somatotropin and IGF-1 (r = 0.05, P > 0.76) or between progesterone and somatotropin (r = 0.04, P > 0.631, but there was a significant negative correlation between progesterone and IGF-1 (r = -0.26, P I 0.01). DISCUSSION The significant increase in the number of corpora lutea in superovulated heifers administered 500 mg of rBST depot on Day 11, coincidentally with the first FSH injection, is consistent with our previous observation that multiple (twice-daily) 20-mg injections of rBST during FSH treatment improves the superovulatory response (15). The tendency toward an increased number of corpora lutea in the animals given the rBST depot at Day 4 is similar to the moderate, nonsignificant increase observed in cows given a 640-mg depot of rBST, 5 days before treatment with PMSG (16). These observations suggest that rBST, either directly or through IGF-1, improves the ability of subordinate follicles to develop in response to exogenous gonadotrophins in heifers, as has been shown in women (9-14). However, the fact that treatment with the depot on Day 15 (at the time of AI) had no effect on the number of corpora lutea suggests that rBST did not directly affect ovulation or luteinization. Treatment with the rBST depot had no statistically significant effect on the total number of embryos or on the number of transferable embryos, consistent with our previous observations using twice-daily injections of rBST (15), but all 3 rBST-treated groups yielded approximately one more transferable embryo per collection than did the controls. Moreover, the overall percentage of transferable embryos was significantly increased (P I 0.01) in the animals administered the rBST depot on Day 4. This may suggest that somatotropin or IGF-1 is particularly important for the early development of the antral follicle and the oocyte. Gong et al. (21) reported that daily injections of rBST over 2 estrous cycles doubles the number of small antral follicles in otherwise untreated heifers, suggesting that somatotropin or IGF-1 can affect follicular development at an even earlier stage. Although our experiment was designed to test only the effects of rBST on the superovulatory response, the inverse relationship that we observed between plasma concentrations of progesterone and IGF-1 in the control animals was striking, and perhaps of fundamental interest. This relationship may suggest that the secretion of IGF-1 is related to one or more factors
associated with ovarian development in the superovulated heifer, but that mechanism is not clear. Plasma concentrations of IGF-1 decrease significantly within 24 hours after ovariectomy in beef-cows (22) and cross-breed heifers (23), and increase significantly within 14 hours after treatment of ovariectomized cows with estradiol (22). It is therefore possible that the inverse relationship between IGF-I and progesterone reflected the changes in circulating estradiol during follicular development. However, further investigation is required to determine the exact mechanism and physiological significance of changes in 1GF-l secretion during the estrous cycle in cattle. In conclusion, treatment of superovulated heifers with a 500-mg depot injection of rBST on Day 11, coincidentally with the first FSH injection, produced a significant increase in the ovulatory response. Treatment with the depot on Day 4,7 days before the first FSH injection, tended to increase the ovulatory response and significantly increased the percentage of transferable In the control animals, plasma concentrations of IGF-1 were embryos. negatively correlated with progesterone, but were statistically unrelated to somatotropin, suggesting that IGF-1 secretion in the superovulated heifer may be related to some factor associated with follicular development, possibly to circulating estradiol.
1.
Hammond, J.M., Mondschein, J.S., Samaras, S.E., Smith, S.A. and Hagen, D.R. The ovarian insulin-like growth factor system. J. Reprod. Fertil. 43 (Suppl.):199-208 (1991).
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Dorrington, J.H., Bendell, J.J., Chuma, A. and Lobb, D.K. Actions of growth factors in the follicle. J. Steroid Biochem. 27:405-411 (1987).
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Effect of Wilson, E.E., Word, R.A., Byrd, W. and Carr, B.R. superovulation with human menopausal gonadotropins on growth hormone levels in women. J. Clin, Endocrinol. Metab. 73:511-515 (1991).
factors in embryonic
development.
factors
in
early
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Theriogenoiogy
7
Menashe, Y., Lunenfeld, B., Pariente, C., Frenkel, Y. and Mashiach, S. Can growth hormone increase, after clonidine administration, predict the dose of human menopausal hormone needed for induction of ovulation. Fertil. Steril. %:432-435 (1990).
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Oyesen, I’., Moller, Jorgensen,, J.O.L. insulin-like growth women with regular
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Homburg, R., West, C. and Jacobs, H.S. Controlled trial of co-treatment with growth hormone and gonadotrophins for induction of ovulation. J. Endocrinol. 119 ~Suppl.):l44 abstr. (1988).
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Blumenfeld, Z. and Lunenfeld, B. The potentiating’ effect of growth hormone on follicle stimulation with human menopausal gonadotropin in a panhypopituitary patient. @:328-331 (1989).
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Matson, P.L., Ibrahim, Z.H.Z., Morris, I.D., Sun, J.G. and Lieberman, B.A. Effect of biosynthetic human growth hormone upon follicular growth in women undergoing ovarian stimulation prior to in-vitro fertilisation and embryo transfer. J. Reprod. Fertil. Abstr. Ser. 4:17 abstr. (1989).
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Homburg, R., West, C., Torresani, T. and Jacobs, H.S. Cotreatment with human growth hormone and gonadotropins for induction of ovulation: a controlled clinical trial. Fertil. Steril. 3:254-260 (1990).
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Homburg, R., West, ‘C., Torresani, T. and Jacobs, H.S. A comparative study of single-dose growth hormone therapy as an adjuvant to gonadotrophin treatment for ovulation induction. Clin. Endocrinol. 32:78X-785 (1990).
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Ibrahim, Z.H.Z., Matson, P.L., Buck, P. and Lieberman, B.A. The use of biosynthetic human growth hormone to augment ovulation induction with buserelin acetate/human menopausal gonadotropin in women with a poor ovarian response. Fertil. Steril. z:202-204 (1991).
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Rieger, D., Walton, J.S., Goodwin, M.L. and Johnson, W.H. The effect of co-treatment with recombinant bovine somatotrophin on plasma progesterone concentration and numbers of embryos collected from superovulated Holstein heifers. Theriogenology %:863-868 (1991).
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Herrler, A., Farries, E. and Niemann, H. A trial to stimulate insulin like growth factor 1 levels to improve superovulatory response in dairy cows. 33:248 abstr. (1990).
J., Moller, N., Christiansen, J.S., Orskov, H. and Growth hormone secretory capacity and serum factor-I levels in primary Infertile, anovulatory menses. Fertil. Steril. 57:97-101 (1992).
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Elsden, R.P., Nelson, L.D. and Seidel, G.E. Superovulating cows with follicle stimulating hormone and pregnant mare’s serum gonadotrophin. Theriogenology 2:17-26 (1978).
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Elsasser, T.H., Rumsey, T.S. and Hammond, A.C. Influence of diet on basal and growth hormone-stimulated plasma concentrations of IGF-1 in beef cattle. a:128-141 (1989).
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Groenewegen, P.P., McBride, B.W., Burton, J.H. and Elsasser, T.H. Effect of bovine somatotropin on the growth rate, hormone profiles and carcass composition of Holstein bull calves. Dom. Anim. Endocrinol. 2:43-54 (1990).
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de Boer, G., Etches, R.J. and radioimmunoasay for progesterone Sci. @:783-786 (1980).
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Gong, J.G., Bramley, T. and Webb, R. The effect of recombinant bovine somatotropin on ovarian function in heifers: follicular populations and peripheral hormones. Biol. Reprod. a:941-949 (1991).
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Richards, M.W., Wettemann, RI’., Spicer, Nutritional anestrus in beef cows: effects ovariectomy on serum luteinizing hormone factor-l. Biol. Reprod. @:961-966 (1991).
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Spicer, L.J., Crow, M.A., Prendiville, D. J., Goulding, D. and Enright, Systemic but not intraovarian concentrations of insulin-like W.J. growth factor-l are affected by short-term fasting. Biol. Reprod. &:920925 (1992).
Walton, J.S. A solid-phase in bovine plasma. Can. J. Anim.
L.J. and Morgan, G.L. of body condition and and insulin-like growth